Characterization of cytotoxicity induced by sphingolipids in multidrug-resistant leukemia cells

doi:10.1016/S0145-2126(98)00107-6

Leukemia Research

Volume 22, Issue 11, November 1998, Pages 1049-1056

https://doi.org/10.1016/S0145-2126(98)00107-6 Get rights and content

Abstract

Certain sphingolipids (SLs) exert fundamental roles in differentiative, growth-inhibitory and apoptotic pathways induced by a number of agents in leukemia cells. Multidrug-resistance (MDR) is a major cause of therapeutic failure in leukemia. SLs are among the diverse substrates for the MDR p-170 glycoprotein drug-efflux pump. We tested the hypothesis that expression of MDR would thereby block the cytotoxicity induced by the SLs sphingosine, sphinganine and N-hexanoyl-sphingosine. An MDR-expressing subline of murine P388 leukemia cells demonstrated an ED₅₀ value ≥2 log₁₀ higher than the parental line in response to doxorubicin. In contrast, the ED₅₀ values for each of the SLs were only approximately 1.5 to two-fold higher in the MDR line than in the parental; induction of DNA damage by SLs was comparable or actually greater in MDR compared to parental cells. Therefore, expression of MDR does not significantly affect the cytotoxic function of these SLs, nor do these SLs likely contribute to MDR.

Introduction

Inherent and treatment-induced chemoresistance is a major cause of therapeutic failure in clinical oncology. Chemoresistance is complex and multifactorial, influenced by cellular and molecular pharmacologic mechanisms, altered enzymatic pathways and expression of certain oncogenes 1, 2, 3. Among the best characterized and most widespread chemoresistance mechanisms in tumor cells, including leukemias [4], is expression of the p-170 multidrug-resistance (MDR) glycoprotein 5, 6, a member of the family of ATP-binding cassette-containing membrane transporters. This drug-efflux pump can sequester [7]or remove drugs from the cell 4, 5, 6to an extent that renders available intracellular drug levels incapable of inducing apoptosis.

Substrates for the MDR P-glycoproteins (P-gps) are as structurally diverse as the agents that demonstrate susceptibility to MDR; the latter include Vinca alkaloids, anthracyclines and epipodophyllotoxins. These substrates are generally characterized only as sharing an amphipathic nature, critical to their molecular binding interaction with the P-gp membrane pump. Some investigators propose that this binding occurs at the cytoplasmic leaflet of the plasma membrane followed by a transmembrane `flipping' of the polar group of the substrate by the P-gp out to the exoplasmic face [8].

Recent studies have demonstrated that both human isoforms of the P-gp, the gene products of mdr-1 and mdr-3, are lipid translocases 8, 9, 10, 11, 12. MDR1 P-gp translocated a wide variety of sphingolipids (SLs) to both apical and basolateral membranes at temperatures ≥20°C; these SLs included the fluorophores N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphingosine-1-phosphocholine (C₆-NBD-sphingomyelin; SM) and C₆-NBD-glucosyl-ceramide, as well as short-chain glucosyl-ceramide analogues [12]. Incubation at 15°C blocked the vesicle-dependent transport of C₆-NBD-SM from the lumen of the Golgi; export of C₆-NBD-glucosyl-ceramide from the cytoplasmic face of the Golgi and endoplasmic reticulum still occurred at this temperature and only in the MDR1 expressing cells, indicating an essential role for the P-gp in this translocation. These SLs were synthesized by the cells from a C₆-NBD-ceramide precursor [13].

These observations of the structural specificity of MDR1 P-gp for SLs raise provocative questions in light of the recently disclosed role of SLs as putative downstream mediators in differentiative, growth-inhibitory and apoptotic pathways induced in tumor cells (reviewed in [14]). The SL ceramide, derived either from catalytic cleavage of sphingomyelin (SM) or via de novo synthesis triggered by exposure to a number of upstream stimuli, including differentiating agents and chemotherapeutic drugs, induces classical patterns of apoptosis in human HL-60 and U937 leukemia cells [15]as well as many other tumor targets; the latter involved the use of exogenously administered cell-permeable ceramide analogues. Similarly, the non-acylated counterpart of ceramide, sphingosine (So), is elevated during phorboid-induced differentiation of HL-60 cells [16]and induces growth-arrest and apoptosis in tumor cells following exogenous administration [17]. Of therapeutic relevance are the observations that So formulations have shown antitumor and antimetastatic activity in pre-clinical tumor models, although their mechanism of action is not unequivocally defined 18, 19, 20, 21.

In light of the activity of MDR1 against SL substrates, we predicted that the P-gp drug-efflux pump would protect cells against apoptosis/cytotoxicity, not only by exporting the upstream, proximal inducer of apoptosis, the chemotherapeutic agent itself, but also by exporting SLs, the distal mediators essential to the apoptotic pathway. The current studies were designed to test the latter aspect of this hypothesis in vitro.

Section snippets

Cell lines

The murine P388 myeloid leukemia cell line and its MDR-expressing counterpart were obtained from Dr. Roman Perez-Soler, Department of Thoracic and Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center. The MDR line was selected from the parental by growth in the presence of increasing concentrations of doxorubicin (Dox) and displayed a stable resistance phenotype in the absence of further selection pressure. These lines were maintained in DMEM/F-12 medium (GIBCO,

Comparison of susceptibility of parental and MDR P388 cells to Dox cytotoxicity

Both parental and MDR P388 cells were cultured for up to 48 h in the presence of Dox, using a dose range established by twofold titrations from 5 μg/ml to ~6 ng/ml. The results from the MTT assay (shown in Fig. 2(A)) indicated that the ED₅₀ for the parental cells was between 2.4 and 4.9 ng/ml, whereas the ED₅₀ for the MDR cells was in the 0.625–1.25 μg/ml range (Fig. 2(B)). Comparison of the survival of parental cells treated with 2.4 ng/ml (74.5±1.3% survival) or 4.9 ng/ml Dox (33.9±1.5%

Discussion and conclusion

Our results demonstrate that the SLs C₆-Cer, So and Sa exert cytotoxic effects in murine P388 leukemia cells irrespective of the expression of MDR in these cell lines. In light of the reported activity of MDR1 as a lipid translocase of broad selectivity and of the effectiveness of lipids with a ceramide backbone as substrates for the P-gp pump in this context 8, 12, we did not anticipate this result. In contrast, MDR3 has not been reported to display drug-pumping activity [24]and has a lipid

Acknowledgements

We thank Kim D. Steck and Douglas A. Weidner for conducting the flow cytometric analyses. These analyses were conducted with support from the Automated Cytometry component of the Cancer Center Support Grant CA 16672. We thank Michael S. Worley of the Department of Scientific Publications for helpful editorial comments.

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Characterization of cytotoxicity induced by sphingolipids in multidrug-resistant leukemia cells

Abstract

Introduction

Section snippets

Cell lines

Comparison of susceptibility of parental and MDR P388 cells to Dox cytotoxicity

Discussion and conclusion

Acknowledgements

Biochim Biophys Acta

Trends Biochem Sci

Cell

Cell

Chem Phys Lipids

Cell

J Biol Chem

Leuk Res

J Biol Chem

J Biol Chem

Cell

Atypical multiple drug resistance in a human leukemic cell line selected for resistance to teniposide (VM-26)

Cancer Res

Combined modalities of resistance in etoposide-resistant human KB cell lines

Cancer Res

Drug resistance modulation in the laboratory and the clinic

Semin Oncol